低毒力病毒CHV1（Cryphonectria hypovirus 1）5’-端3个基因的克隆及板栗疫病生物防治
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摘要
栗疫菌细胞中的低毒力病毒是一类没有衣壳的双链RNA病毒,可以引起寄主栗疫菌的菌落形态变化、分生孢子和色素的产量降低、雌性不育,和漆酶产量与活性降低。更重要的是,低毒力病毒的侵染会导致栗疫菌毒力的下降,这一发现为栗疫病的生物防治提供了一种全新的思路。
目前已经报道了3个CHV1 (Cryphonectria hypovirus 1)的全基因组序列,这三个菌株的序列同源性非常高,然而本实验室对中国CHV1初步的测序结果发现中国的CHV1群体结构更为复杂。为了更好地研究中国的低毒力病毒,本文从更长的序列上分析了CHV1的多样性。首先,利用本实验室已全基因组测序的中国菌株CHV1-CN280,结合已发表的3株CHV1病毒,在序列保守区域设计了特异性的引物。然后基于RT-PCR技术,分4段克隆和测序了来自中国、日本和意大利共14株低毒力病毒的5’-端约3200bp序列。分析了这些序列中的5'-UTR(untranslated region, UTR)、p29、p40和p48的差异,同时利用这些序列分析了低毒力病毒的系统进化关系。
实验结果表明,CHV1的5’-UTR和p29变异较大,根据这两者作的系统进化树较相似。并且发现CHV1-Ja19的p29蛋白在C-端存在自然缺失。p40在3个蛋白中最为保守,所作的系统进化树没有明确的分枝。p48在N-端有较大变异,而C-端则趋于保守。通过测序结果分析发现CHV1两个开放阅读框之间的重叠连接序列也具有多样性。中国北方的群体为八核苷酸序列AUGUAUAA,南方群体为五核苷酸序列UAAUG;在GenBank中的序列检索还新发现欧洲菌株中也有八核苷酸连接方式。比较不同蛋白的系统进化树发现,CHV1中普遍存在遗传重组现象。例如,CHV1-CN431、CHV1-CN238和CHV1-CHR011的5’-UTR和p29的N-端与中国北方菌株的亲缘关系更近,p48却与南方菌株的亲缘关系更近,CHV1-A1 30的情况则刚好相反；两个日本菌株的5’-UTR和p29与中国南方菌株亲缘关系非常近,但是p48的亲缘关系却很远,似乎有其它的来源。最后对所测序列的全长作系统进化分析可以看出,欧洲的CHV1病毒与中国北方的低毒力病毒群体处于一个分枝内,表明二者亲缘关系更近。
本文另外报道了在浙江省遂昌县进行栗疫病生物防治所取得的阶段性成果。低毒力菌株在田间的定殖和传播较为困难,但是通过处理自然发病的病斑,可以将低毒力病毒引入板栗林中的栗疫菌群体,而且营养体不亲和性在田间对低毒力病毒的传播影响有限。考虑到中国栗疫菌复杂的遗传多样性,这对于中国果疫病的生物防治来说是个好消息,但是影响中国生物防治成败的其它因素还有待探索。
Hypoviruses are a group of unencapsidated, cytoplasmically replicating double-stranded RNA viruses which can alter host biological processes, resulting in modified colony morphology, suppressed pigmentation, reduced asexual production, loss of female fertility, and reduced laccase production and activity. But most of all, hypovirus will reduce virulence of Ciyphonectria parasitica, of which the discovery presented a new avenue for biological control of chestnut blight.
To date three European isolates of Cryphonectria hypovirus 1 (CHV1) which have been submitted to GenBank share very high homology, compared to various divergence of Chinese CHV1 populations according to a group of short sequences reported by co-workers of our laboratory. To further look into the biological diversity of CHV1 in China, we have analyzed a series of longer sequences. Taking the usage of CHV1-CN280 which has been sequenced throughout the genome in our lab, and the other three European isolates, we designed specific primers within the conserved region. And then 14 isolates from China, Japan and Italy were cloned and sequenced in the 5'-proximal region about 3200bp by four split segments, based on the technique of reverse transcript PCR (RT-PCR). Following which the variation and phylogenesis of 5'-UTR, p29, p40, p48 and their orderly spliced sequences were analyzed.
We now report the similar diversity of 5'-URT (untranslated region) and protein p29 compared to the more conserved p40, of which the phylogenetic tree shows indistinct branches, while p48 shows great variation in the N-terminal portion and tend to be conserved in the C-terminal region. It is intriguing that a deletion-mutated p29 is discovered in the CHV1-Jal9 isolate. The overlapping codons between the two open reading frames (ORF) of CHV1 are also found to be varied, with that all of North Chinese isolates contain octanucleotide AUGUAUAA, yet South Chinese and European CHV1 isolates incline to share pentanucleotide UAAUG. Besides. three CHV1 isolates from Europe comprising octanucleotide are also discovered with the help of blast in GenBank. In the work of phylogenetic analysis, we have revealed extensive occurrence of RNA recombination among CHV1 populations. For example, the N-terminal region of 5'-UTR and p29 of CHV1-CN431, CHV1-CN238 and CHV1-CHR011 are genetically closer, but CHV1-A 130 in opposite result; and the closer genetic relationship between two Japanese isolates and South Chinese isolates within 5'-UTR and p29 is observed, yet deviate from each other in p48 segment, implying unknown genomic resources for Japanese CHV1. Moreover. European CHV1 are proposed closer relationship with North Chinese CHV1.
It is reported in addition about recent achievement in biological control of chestnut blight in Suichang County, Zhejiang province of China. Hypovirulent strains of C. parasitica will encounter challenge for colonization and spread in the field. However, when inoculated in the edge of chestnut blight cankers. CHV1 can be transmitted into natural populations in the field, regardless of vegetative incompatibility. Given to genetic diversity of Chinese C. parasitica, it is good news for biological control of chestnut blight in China, and other more associated factors need to be researched.
目前已经报道了3个CHV1 (Cryphonectria hypovirus 1)的全基因组序列,这三个菌株的序列同源性非常高,然而本实验室对中国CHV1初步的测序结果发现中国的CHV1群体结构更为复杂。为了更好地研究中国的低毒力病毒,本文从更长的序列上分析了CHV1的多样性。首先,利用本实验室已全基因组测序的中国菌株CHV1-CN280,结合已发表的3株CHV1病毒,在序列保守区域设计了特异性的引物。然后基于RT-PCR技术,分4段克隆和测序了来自中国、日本和意大利共14株低毒力病毒的5’-端约3200bp序列。分析了这些序列中的5'-UTR(untranslated region, UTR)、p29、p40和p48的差异,同时利用这些序列分析了低毒力病毒的系统进化关系。
实验结果表明,CHV1的5’-UTR和p29变异较大,根据这两者作的系统进化树较相似。并且发现CHV1-Ja19的p29蛋白在C-端存在自然缺失。p40在3个蛋白中最为保守,所作的系统进化树没有明确的分枝。p48在N-端有较大变异,而C-端则趋于保守。通过测序结果分析发现CHV1两个开放阅读框之间的重叠连接序列也具有多样性。中国北方的群体为八核苷酸序列AUGUAUAA,南方群体为五核苷酸序列UAAUG;在GenBank中的序列检索还新发现欧洲菌株中也有八核苷酸连接方式。比较不同蛋白的系统进化树发现,CHV1中普遍存在遗传重组现象。例如,CHV1-CN431、CHV1-CN238和CHV1-CHR011的5’-UTR和p29的N-端与中国北方菌株的亲缘关系更近,p48却与南方菌株的亲缘关系更近,CHV1-A1 30的情况则刚好相反；两个日本菌株的5’-UTR和p29与中国南方菌株亲缘关系非常近,但是p48的亲缘关系却很远,似乎有其它的来源。最后对所测序列的全长作系统进化分析可以看出,欧洲的CHV1病毒与中国北方的低毒力病毒群体处于一个分枝内,表明二者亲缘关系更近。
本文另外报道了在浙江省遂昌县进行栗疫病生物防治所取得的阶段性成果。低毒力菌株在田间的定殖和传播较为困难,但是通过处理自然发病的病斑,可以将低毒力病毒引入板栗林中的栗疫菌群体,而且营养体不亲和性在田间对低毒力病毒的传播影响有限。考虑到中国栗疫菌复杂的遗传多样性,这对于中国果疫病的生物防治来说是个好消息,但是影响中国生物防治成败的其它因素还有待探索。
Hypoviruses are a group of unencapsidated, cytoplasmically replicating double-stranded RNA viruses which can alter host biological processes, resulting in modified colony morphology, suppressed pigmentation, reduced asexual production, loss of female fertility, and reduced laccase production and activity. But most of all, hypovirus will reduce virulence of Ciyphonectria parasitica, of which the discovery presented a new avenue for biological control of chestnut blight.
To date three European isolates of Cryphonectria hypovirus 1 (CHV1) which have been submitted to GenBank share very high homology, compared to various divergence of Chinese CHV1 populations according to a group of short sequences reported by co-workers of our laboratory. To further look into the biological diversity of CHV1 in China, we have analyzed a series of longer sequences. Taking the usage of CHV1-CN280 which has been sequenced throughout the genome in our lab, and the other three European isolates, we designed specific primers within the conserved region. And then 14 isolates from China, Japan and Italy were cloned and sequenced in the 5'-proximal region about 3200bp by four split segments, based on the technique of reverse transcript PCR (RT-PCR). Following which the variation and phylogenesis of 5'-UTR, p29, p40, p48 and their orderly spliced sequences were analyzed.
We now report the similar diversity of 5'-URT (untranslated region) and protein p29 compared to the more conserved p40, of which the phylogenetic tree shows indistinct branches, while p48 shows great variation in the N-terminal portion and tend to be conserved in the C-terminal region. It is intriguing that a deletion-mutated p29 is discovered in the CHV1-Jal9 isolate. The overlapping codons between the two open reading frames (ORF) of CHV1 are also found to be varied, with that all of North Chinese isolates contain octanucleotide AUGUAUAA, yet South Chinese and European CHV1 isolates incline to share pentanucleotide UAAUG. Besides. three CHV1 isolates from Europe comprising octanucleotide are also discovered with the help of blast in GenBank. In the work of phylogenetic analysis, we have revealed extensive occurrence of RNA recombination among CHV1 populations. For example, the N-terminal region of 5'-UTR and p29 of CHV1-CN431, CHV1-CN238 and CHV1-CHR011 are genetically closer, but CHV1-A 130 in opposite result; and the closer genetic relationship between two Japanese isolates and South Chinese isolates within 5'-UTR and p29 is observed, yet deviate from each other in p48 segment, implying unknown genomic resources for Japanese CHV1. Moreover. European CHV1 are proposed closer relationship with North Chinese CHV1.
It is reported in addition about recent achievement in biological control of chestnut blight in Suichang County, Zhejiang province of China. Hypovirulent strains of C. parasitica will encounter challenge for colonization and spread in the field. However, when inoculated in the edge of chestnut blight cankers. CHV1 can be transmitted into natural populations in the field, regardless of vegetative incompatibility. Given to genetic diversity of Chinese C. parasitica, it is good news for biological control of chestnut blight in China, and other more associated factors need to be researched.
引文
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